Abstract

Growth arrest–specific 1 (Gas1) plays a critical role in growth suppression. Previous study indicated that Gas1 was closely associated with survival in patients with colorectal cancer; however, the underlying molecular mechanism remains unclear. In this study, we sought to determine the role of Gas1 in tumorigenesis and metastasis, and elucidate the possible mechanism. First, Gas1 was determined as a negative regulator of oncogenesis and metastasis in colorectal cancer. Mechanistically, Gas1 negatively regulated the aerobic glycolysis, a process that contributed to tumor progression and metastasis by providing energy source and building blocks for macromolecule synthesis. To further consolidate the role of Gas1 in glycolysis, the impact of Gas1 in the transcription of key glycolytic enzymes for glucose utilization was examined. As expected, GLUT4, HK2, and LDHB exhibited a decreased expression pattern. Consistent with this observation, an in vivo subcutaneous xenograft mouse model also confirmed the hypothesis that Gas1 is a negative regulator of glycolysis as reflected by the decreased 18FDG uptake in PET/CT system. Moreover, Gas1 negatively regulated the AMPK/mTOR/p70S6K signaling axis, a well-established cascade that regulates malignant cancer cell behaviors including proliferation, metastasis, and aberrant cancer metabolism. In the end, it was determined that Gas1 is a transcriptional target of FOXM1, whose role in colorectal cancer has been widely studied. Taken together, these studies establish Gas1 as a negative regulator in colorectal cancer.

Introduction

Colorectal cancer is one of the leading malignancies worldwide and is the third cause of death in cancer patients (1). Although significant progress has been made in diagnosis and treatment of colorectal cancer, invasion, metastasis, and recurrence of the disease are still challenging (2). Hence, there is an urgent need to better understand the genetic and biological characteristic of colorectal cancer, which will improve the efficacy of the treatment of this disease, including surgical techniques, chemotherapy methods, and follow-up strategies.

Tumor invasion and metastasis are parts of a complicated process in which the tumor grows, then detaches from the primary site and metastasizes to a distant organ. Previous research has demonstrated that epithelial–mesenchymal transition (EMT) plays a key role in the early process of the metastasis of cancer cells. This process involves the acquisition of the expression of mesenchymal molecules, such as vimentin and N-cadherin, together with the loss of epithelial cell adhesion molecules such as E-cadherin (3, 4).

Recent study indicated that metabolic reprogramming plays critical roles during the EMT process, and provides metabolic advantage for EMT cells (5, 6). Normally differentiated cells rely primarily on the oxidation of pyruvate in the mitochondria to generate energy for cellular physiology; however, even with sufficient oxygen, rapidly growing cancer cells rely on aerobic glycolysis to generate energy. This phenomenon is termed as the Warburg effect. The Warburg effect not only provides cancer cells with ATP and nutrients, but also creates an acidic environment that leads to destruction of extracellular matrix and facilitates metastasis. Therefore, identifying key players synergistically regulates the metastasis and glycolysis will provide powerful strategies in the diagnosis and treatment for colorectal cancer (7).

Aberrations of protein-coding genes, including both oncogenes and tumor suppressive genes have been widely accepted to play critical roles in process of colorectal cancer. Previously, our studies showed that growth arrest–specific protein 1 (Gas1) could contribute to predicting metastasis or recurrence in stage II and III colorectal cancer (8). However, the underlying mechanisms that Gas1 contributed to colorectal cancer oncogenesis and metastasis remain elusive. Hence, in this study, we performed a series of in vitro and in vivo studies and demonstrated Gas1 as a tumor suppressor in colorectal cancer. Mechanistically, Gas1 negatively regulated the EMT process, and was indicated as a negative regulator of glycolysis. Further clinical and pathologic analyses demonstrated that Gas1 expression level was negatively associated with SUVmax value reflected by PET/CT imaging, supporting the notion of Gas1 as a negative regulator in vivo. In the end, we determined that Gas1 negatively regulated the AMPK/mTOR/p70S6K signaling axis, and Gas1 itself was a downstream target of FOXM1, a well-established player in colorectal cancer.

Materials and Methods

Patient information and tissue specimens

For PET/CT and Warburg study, the first study cohort included 71 colorectal cancer patients who underwent radical surgery between January 2008 and December 2012 at Fudan University Shanghai Cancer Center (FUSCC). Preoperative 18F-FDG PET/CT examination and histopathology confirmation of the presence of colorectal adenocarcinoma were conducted in all patients. The demographic and clinical characteristics of the patients are summarized in Supplementary Table S1.

For tissue microarray (TMA) based IHC study, colon cancer tissues were obtained from 185 patients who underwent initial radical surgery, including 24 cases at stage I, 81 at stage II, and 80 at stage III. All the patients had a histologic diagnosis of colon cancer. Detailed clinical characteristics of the patients are summarized in Supplementary Table S2.

None of these patients included in the study had received neoadjuvant therapy. All the subjects involved in this study provided written informed consent. This project was approved by the Ethics Committee of FUSCC.

Construction of the TMA and IHC staining

Construction of the TMA and IHC staining were performed as described previously (9, 10). Gas1 and FOXM1 anti-human rabbit polyclonal antibodies were used at a dilution of 1:100 (AP11869a; Abgent) and 1:50 (sr-500; Santa Cruz Biotechnology), respectively; PBS was used alternately as negative control. All immunostainings were independently evaluated by two pathologists and a consensus justification based on discussion was recorded. For clinicopathologic correlation analysis, we used a four-tiered scoring system (negative to 3+), which took into account the percentage of positive cells and staining intensity (8, 11). Gas1 was positively stained at cytoplasm and membrane whereas FOXM1 was nucleolus stained. We separately interpreted 0 and 1+ as “low expression,” whereas 2+ and 3+ as “strong expression.”

Cell culture

The human colon cancer cell lines HT-29, HCT116, SW480, SW620, RKO, Colo205, Ls174T, and LoVo were originally obtained from the ATCC. The cells were cultured in RPMI1640 medium containing 10% FBS in a humidified 37°C incubator supplemented with 5% CO2.

Plasmids and the establishment of stable transfection cell lines

Gas1 full-length cDNA was cloned from HCT116 cDNA using primers: 5′- ATGGTGGCCGCGCTGCTGGGC -3′ (forward primer) and 5′- CTAAAAGAGCGGCCCAAGCAG -3′ (reverse primer). PCR product was cloned into PCMV-N-flag vector, and then FLAG-tagged Gas1was cloned into pCDH-CMV-MCS-EF1-Puro vector to generate pCDH-Gas1 construct. pLKO.1 TRC cloning vector (Plasmid10878; Addgene) was used to generate constructs expressing shRNAs against Gas1. The 21bp shRNA target against Gas1 were 5′-GGGCTGTCTATTAGCATATTT-3′ (1#) and 5′-GCCATGTATGAAAGTCTC-3′ (2#), respectively. pLKO.1-scramble shRNA (Plasmid1864; Addgene) with limited homology with any known sequences in the human was used as a negative control. For establishment of stable cells that expressed Flag-Gas1, HCT116 and SW480 were transfected with the pCDH-Gas1 expression vector and the control vector. For establishment of knockdown stable cells, RKO and HT29 were transfected with the pLKO.1-shGas1 expression vector and pLKO.1-scramble. Transfected cells were selected using puromycin after the cells were transfected with expression/knockdown vector or control plasmids. These stable cells were all used for functional studies as below in the text.

Cell proliferation and clonogenic assay

Cell proliferation was assessed by CCK8 assay as described previously (10). To determine clonogenic ability, 200 cells were transplanted in each well of a 6-well dish and allowed to grow for 14 days to form colonies. Cells were fixed with methanol and stained with 0.1% crystal violet. All the visible colonies were calculated manually.

In vitro migration and invasion assays

Migration and invasion assay were performed using a Transwell system (Costar) according to manuals. Chambers were incubated at 37°C for 24 hours, and three duplicates were prepared for each group. Successfully translocated cells were fixed and then stained with 0.2% crystal violet. The total cell numbers of five random visual fields were counted, and the average was calculated.

E-cadherin and vimentin immunofluorescence

Cells were grown on coverslips, fixed in 4% paraformaldehyde for 20 minutes, incubated in a blocking buffer (1% BSA and 0.25% Triton X-100 in PBS; pH 7.4), and probed with an E-cadherin antibody or vimentin antibody, then cells were incubated with Alexa Flour 594 TgG donkey anti-rabbit (1:500; Invitrogen) for an hour at room temperature. To detect nuclei, cells were costained with DAPI. Fluorescence images were photographed with a confocal microscopy.

Glycolysis analysis

Glucose Uptake Colorimetric Assay Kit (Biovision) and Lactate Colorimetric Assay Kit (Biovision) were purchased to examine the glycolysis process in colon cancer cells according to the manufacturer's protocol. Real-time PCR was performed to test expression of glycolytic enzymes. All reactions were run in triplicate.

Cell apoptosis rate analysis

Cells stably transfected with PCDH-Gas1 and Gas1-shRNA were used for this analysis. For apoptosis rate analysis, cells were incubated with Annexin V-FITC (BD Biosciences) and propidium iodide for 10 minutes at room temperature in the dark. After staining, the cells were analyzed using a flow cytometer (CYTOMICS FC 500; Beckman Coulter).

RNA isolation and qRT-PCR analysis

Total RNA from the tissues and cells was extracted using TRIzol reagent (Invitrogen). RNA quality and concentration were determined using the Nanodrop 2000 system (Thermo Fisher Scientific). The expression status of target genes and β-actin were determined by qRT-PCR using an ABI 7900HT Real-Time PCR system (Applied Biosystems) using a Power SYBR Green PCR Master Mix (Invitrogen). All reactions were run in triplicate. All RT-PCR primers were displayed in Supplementary Table S3.

Luciferase assays

For the luciferase assays, the Gas1 promoter was cloned into the pGL3 basic vector (Promega). Then, HCT116 and RKO cells (8 × 103 cells/well) were cultured in 94-well plates and cotransfected with the pGL3-Gas1, pGL3-control, or pGL3-Gas1(mutated), pcDNA3.1-FOXM1/pcDNA-control, and Renilla plasmid using Lipofectamine 3000 (Invitrogen). Forty-eight hours after transfection, cells were lysed using 20 μL of passive lysis buffer. Next, a dual-luciferase assay was carried out as directed by the manufacturer (Promega). The ratio of firefly to Renilla luciferase activity was used to express luciferase activities. All experiments were performed in triplicate. Data are represented as mean ± SD.

Chromatin immunoprecipitation assay

HCT116 and RKO cells were prepared for a chromatin immunoprecipitation (ChIP) assay using ChIP Assay Kit (Millipore) according to the manufacturer's protocol. The resulting precipitated DNA samples were analyzed using PCR to amplify two potential binding region of the Gas1 promoter with the primers 1# 5′-GTGGTGATCAAGACCCAAAGACAG-3′(forward primer) and 5′-TAAGGAGGCTCGGATATGCAGCCC-3′ (reverse primer) and 2# 5′-GGAGAAAGGAGAAAGCGGGCAGGC-3′ (forward primer) and 5′-TGGCTTCACTCGGCGGCAGCTTC-3′ (reverse primer). The PCR products were resolved electrophoretically on a 1.5% agarose gel and visualized using Goodview staining.

Xenografted nude mice model

To evaluate in vivo tumorigenesis, five nude mice (male, 4–8 weeks old Balb/C athymic nude mouse) were prepared for HCT116 cells implantation transfected with pCDH-Gas1 or pCDH-vector and RKO cells transfected with PLKO.1-ShGas1 or PLKO.1-scramble. Cells were injected subcutaneously into the right/left forelimbs of nude mice. After 4 weeks, all the injected mice were euthanatized. Tumor xenografts were harvested and weighted. Tumor volume (TV) was calculated weekly for 4 weeks according to the formula: TV (mm3) = length × width2 × 0.5. All animal experiments were performed according to guidelines for the care and use of laboratory animals and were approved by Institutional Animal Care and Use Committee of Fudan University.

Statistical analysis

Data were analyzed using SPSS 21.0 statistical package (SPSS). Based on requirements, either the χ2 or Fisher exact test was applied to assess the correlations between gene expression and various histopathologic features. The Transwell and CCK8 results were analyzed by one-way ANOVA or independent sample t test. A P value < 0.05 was considered statistically significant.

Results

Gas1 inhibited the viability of colon cancer cells in vitro

To assess the role of Gas1 in colon cancer viability and tumorigenic potential, we first examined the endogenous expression level of Gas1 in six colon cancer cells (Supplementary Fig. S1), and then used lentivirus-mediated overexpression of Gas1 in HCT116 and SW480 cells (which exhibited the lowest endogenous Gas1 expression) and silencing of Gas1 in HT29 and RKO cells (which exhibited the highest endogenous Gas1 expression). Overexpression and knockdown efficiency were verified by RT-PCR and Western blotting (Fig. 1A and B). The effect of Gas1 on tumor cell growth was measured by CCK-8 assay and the results demonstrated that knockdown of Gas1 significantly enhanced cells proliferation, whereas overexpression of Gas1 decreased the cell viability with statistical significance (P < 0.05; Fig. 1C). Next, we observed a significant increase in colony formation capacity in cells transfected with Gas1-shRNA. Conversely, there was apparent reduction in colony formation ability in pCDH-Gas1–transfected cells (P < 0.05; Fig. 1D). These observations collectively suggest that Gas1 expression may regulate cell cycle or apoptosis on tumor growth. Gas1 is a cell-cycle arrest protein. Then, we proceeded to use flow cytometer to investigate apoptosis, and found that knockdown of Gas1 reduced cell apoptosis, whereas overexpression of Gas1 induced apoptosis (P < 0.05; Fig. 1E), suggesting that Gas1 regulated the viability of colon cancer cell through both G1 phase arrest and cell apoptosis.

To assess the role of Gas1 on migration and invasion of colon cancer cells, Transwell migration and invasion assays were performed in Gas1 overexpressed or silenced colon cancer cells. The results demonstrated that downregulation of Gas1 expression strongly promoted the migration of RKO and HT29 cells, whereas forced exogenous expression of Gas1 attenuated the migration capacity of HCT116 and SW480 cells (Fig. 1F). This result was also confirmed by invasion assay (Fig. 1G). Because of low invasion ability, SW480 and HT29 were not used for Transwell invasion analysis.

Gas1 inhibited the EMT process of colon cancer cells

To determine whether it was EMT that mediated the inhibitory effect of Gas1 on migration and invasion, we examined the expression of several EMT markers. As expected, overexpression of Gas1 increased the expression level of E-cadherin and decreased the levels of vimentin, N-cadherin, and Snail, whereas knockdown of Gas1 reduced the expression level of E-cadherin and increased the levels of vimentin, N-cadherin, and Snail (Fig. 2A). These results were further confirmed by immunofluorescence (Fig. 2B).

Gas1 inhibits EMT in colon cancer. A, Western blot analysis of phenotypic markers including E-cadherin, N-cadherin, Vimentin, and Snail in Gas1 overexpression and knockdown cells. β-Actin was used as control. B, immunofluorescence analysis of phenotypic marker including E-cadherin, Vimentin. The red signal represents the staining of corresponding protein, and the blue signal represents the nuclear DNA staining by DAP.

Effects of Gas1 on aerobic glycolysis in colon cancer cells in vitro

It is well accepted that tumor formation and progression require glucose metabolism transformation accordingly. Cancer cells exhibit a shift of glucose metabolism to less efficient glycolytic pathways in response to regional hypoxic stress. Under such stress, cancer cells rely on glycolysis to fuel its malignant properties (5, 12, 13). To determine the effect of altered Gas1 expression on aerobic glycolysis in colon cancer cells, we calculated the glucose utilization, and lactate concentrations of the stably transfected cells. Overexpression of Gas1 strongly decreased the glucose utilization, and lactate concentrations in HCT116 and SW480 cells, whereas knockdown of Gas1 expression increased the glucose utilization, and lactate concentrations in RKO and HT29 cells (Fig. 3A). Glycolysis is a multistep enzymatic reaction involved with a series of rate-limiting enzymes (Fig. 3B). To assess the effect of Gas1 on the expression of the rate-limiting enzymes that involved in glycolysis, we carried out qRT-PCR to examine the transcriptional levels of these enzymes. As shown in Fig. 3C and D, both mRNA and protein levels of GLUT4, HK2, and LDHB were significantly reduced in Gas1 overexpression cells, whereas their expressions were significantly increased in Gas1-silenced cells. Taken together, these results validated Gas1 as a negative regulator of glycolysis.

Gas1 is negatively associated with cancer cell growth, tumorigenesis, and Warburg effect in vivo. A1, B1, representative photographs of animals during the course of study. Colon cancer cells were paired subcutaneously injected into forelimbs in nude mice. The left sides were HCT116-Gas1 in A1 and RKO-scramble in B1, whereas the right sides were HCT116-vector in A1 and RKO-shGas1 in B1. Tumors weight and tumor volumes were measured on the indicated days. A2, B2, representative photographs of PET/CT of animals. All mice in A1 and B1 underwent evaluation with 18F-FDG PET/CT scan (laying facedown) before sacrificed. The values of SUVmax were higher in HCT116-vector and RKO-scramble group than paired groups (P < 0.05). A3, B3, immunohistochemical study showed weaker LDHB, GLUT4, HK2, and Ki67 staining in HCT116-Gas1 and RKO-shGas1 groups than their control groups. C, patients with high Gas1 expression always exhibited low SUVmax and low GLUT4, LDHB, and HK2 expression levels, whereas weak Gas1 expression was accompanied with high SUVmax and high GLUT4, LDHB, and HK2 expression.

High uptake of 18F-FDG by tumors has been suggested to be a reflection of the Warburg effect, and the PET/CT imaging system was developed on the basis of the glycolysis thesis as a powerful diagnostic means. All mice underwent evaluation with 18F-FDG PET/CT scan before being sacrificed. The results showed that the SUVmax were higher in HCT116-Vector and RKO-shGas1 group than that of their paired group (Fig. 4A2 and B2). IHC staining also indicated that the expression levels of GLUT4, HK2, and LDHB were lower in Gas1-overexpressed HCT116 cells (Fig. 4A3). Conversely, there were higher GLUT4, HK2, and LDHB expression levels in tumor samples derived from Gas1-silenced RKO cells (Fig. 4B3). These results were also confirmed in a cohort of patients with colon cancer. Patients with high Gas1 expression always exhibited low SUVmax and low GLUT4, HK2, and LDHB expression levels, whereas low Gas1 expression group was accompanied with high SUVmax and high GLUT4, HK2, and LDHB expression. The difference were of statistical significance (Fig. 4C and Supplementary Table S1; P < 0.05).

Gas1 is directly regulated by the transcription factor FOXM1

To explore the transcriptional regulation of Gas1 expression in colorectal cancer, we analyzed transcription factor binding sites spanning the 2.0 kb upstream of transcription starting site. Two Forkhead box family transcription factor binding elements (AAACAA) were identified (Fig. 6A). FOXM1 was selected for further investigation due to its well-established role in colorectal cancer (17, 18). To validate the hypothesis, we first tested the impact of FOXM1 on Gas1 transcription in colon cancer cells and indicated FOXM1 as a negative regulator of Gas1 expression (Fig. 6B and C). Then, we obtained a luciferase reporter construct (pGL3–Gas1–Luc) containing a segment of the human Gas1 promoter and examined the effect of FOXM1 on the promoter activity. Dual luciferase assay indicated that FOXM1 inhibited the Gas1 promoter activity in a dose-dependent manner (Fig. 6D). ChIP assay was performed to demonstrate that FOXM1 occupied the promoter region of Gas1 spanning from −415 to −424 (Fig. 6E). To confirm that this site mediated the Gas1 response to FOXM1, mutations of the selected sequence were introduced by changing the sequence (TTTGTTTGTT) to (CCCACCCACC), which completely abolished the putative responsive site. This mutated version of the Gas1 promoter was cloned into the pGL3-basic-luciferase reporter. A dual-luciferase reporter assay showed that mutation of the putative FOXM1-binding site in the Gas1 promoter completely abolished the FOXM1 responsiveness of the construct (Fig. 6F), demonstrating that the 5′-TTTGTTTGTT-3′ site within the Gas1 promoter mediated the FOXM1 response. TMA-based IHC staining showed a significant reverse association of FOXM1 and Gas1 expression in colon cancer patients (Fig. 6G and Supplementary Table S2). Taken together, these findings suggested transcription factor FOXM1 as a functional regulator of Gas1 in colon cancer.

Gas1 is directly regulated by the transcription factor FOXM1. A, two related sequence within a 2000 bp amplicon in the Gas1 promoter as a putative responsive element able to bind FOXM1 protein. B and C, levels of Gas1, and FOXM1 were determined by Western blot analysis and RT-PCR. HCT116 and RKO cells were transfected with FOXM1 and an empty vector for 48 hours. FOXM1 could significantly decrease Gas1 expression in both mRNA and protein levels (*, P < 0.05). D, luciferase activity of Gas1-luc construct after transfection of FOXM1 plasmid in HCT116 and RKO cells. Luciferase activities were dose dependently decreased after being cotransfected with FOXM1 than with control vector (*, P < 0.05). E, FOXM1 directly bound to the promoter of Gas1 (Site 1#). ChIP assay was performed in HCT116 and RKO cells transfected with a vector expressing FOXM1. RT-PCR was performed using primers specific for site 1# and 2# of the Gas1 promoter (*, P < 0.05; **, P > 0.05). F, mutation of the putative FOXM1-binding site in the Gas1 promoter completely abolished the FOXM1 responsiveness of the construct (*, P > 0.05). G, Gas1 expression was inversely correlated with FOXM1 in patients with colorectal cancer. The expression levels of Gas1 and FOXM1 in 185 patient's samples were determined using IHC. The representative imagines were shown that IHC staining of Gas1 and FOXM1 in colorectal cancer (P < 0.001).

Discussion

It has been reported that metastasis occurs in nearly 50% colorectal cancer patients after curative colectomy, which is the major cause of death in colorectal cancer patients (19). Studies of prognosis for patients with colorectal cancer and of prognostic factors to predict the risk of metastasis for individual colorectal cancer patients are intriguing and could affect clinical practice. Established biomarkers such as KRAS, BRAF, and EGFR have already been proven to play significant roles in prognosis and selection of patients for personalized therapy. As such, there has been a great deal of effort to improve the care of patients and understand the biology of colorectal cancer. Our previous study has shown that Gas1 may serve as a novel prognostic biomarker involved in the pathogenesis and metastasis of colon cancer (8). However, the underlying mechanism of Gas1 still remains elusive. To this end, we sought to identify the role of Gas1 in tumorigenesis and metastasis and provide the possible mechanism. Accumulating studies have reported that Gas1 are low expressed in various cancers and regulated cell growth arrest and apoptosis (20–23). Two recent studies reported that Gas1 expression was associated with drug resistance in non–small cell lung cancer (24) and gastric cancer (25). Gas1 also was identified as a novel melanoma metastasis suppressor gene (26). In consistent with these observations, we confirmed that Gas1 could also regulated cell growth and apoptosis in colorectal cancer. Importantly, we found Gas1 strongly correlated with invasion ability of colorectal cancer by inhibiting EMT and Warburg effect.

EMT is considered to be critical for invasive and metastatic progression in cancer. The process of EMT is associated with the downregulation of epithelial markers and aberrant upregulation of mesenchymal markers. These processes are initiated by zinc finger transcriptional repressors such as Snail, which suppresses E-cadherin expression (27). In this study, we provided evidence that Gas1 was a critical negative regulator of EMT and thereby inhibited the progression and metastasis of colorectal cancer. Knockdown of Gas1 induced downregulation of epithelial markers and upregulation of mesenchymal markers and Snail. Moreover, overexpression of Gas1 resulted in diminished invasion and metastasis of colon cancer cells accompanied with upregulation of epithelial markers and downregulation of mesenchymal markers. Furthermore, in TMA-based IHC study, we found that Gas1 expression was negatively correlated with advanced tumor stage (Supplementary Table S2).

The Warburg effect, a hallmark of cancer cells, has been highlighted in recent decades (7). Coding and noncoding genes may regulate a number of metabolic enzymes, and the aberrantly expressed components might provide a growth advantage for cancer cells. The vast majority of studies on metabolic reprogramming have been performed in the setting of neoplastic transformation. Considerably, little is known about metabolic reprogramming in the context of metastatic transformation. Some studies have pointed out that metastasis is closely related to metabolism transformation. In triple-negative breast cancer, loss of FBP1 by Snail-mediated epigenetic repression provides metabolic advantage for highly metastatic cells (28). However, the correlation between metabolism and metastasis in colorectal cancer has seldom been reported. Because of the role of Gas1 in proliferation and metastasis, we questioned whether the impact was the result of metabolism transformation, because metabolism not only provided cancer cells with energy supply and need for macromolecule synthesis but also created an acidic environment that facilitates breakdown of extracellular matrix that facilitates metastasis. As expected, knockdown of Gas1 increased glucose uptake and lactate secretion, whereas overexpression of Gas1 decreased glucose uptake and lactate secretion. The altered metabolism induced by Gas1 may be required for cancer cell growth and metastasis.

Moreover, we found that Gas1 inhibited EMT and Warburg effect via AMPK activation and mTOR pathway inhibition. AMPK is a sensor of cellular energy level, which is triggered in conditions of low intracellular ATP after various stresses such as hypoxia and nutrient deficiency. AMPK stimulation serves as a metabolic checkpoint to inhibit ATP consuming processes, leading to a stop of cell growth and proliferation (29, 30), and thus reverses EMT (8, 31). Stimulation of AMPK activity requires phosphorylation of the alpha subunit at Thr172 in the activation loop by upstream kinases (29, 32). The fact that Gas1 can induce increasing levels of AMPK phosphorylation at Thr172 supported that Gas1 induces AMPK activity. A large body of evidence has revealed that mTOR signaling is one of the major downstream pathways regulated by AMPK. Recent studies have revealed that AMPK suppresses mTOR activity and the phosphorylation of p70S6K activity (33). The results of this study revealed that Gas1 inhibited EMT and Warburg effect through the AMPK/mTOR/P70S6K pathway. Based on the decisive role of AMPK/mTOR/P70S6K on cell-cycle and nutrient sensing (34–36), in combination with our observation of the inhibitory role of Gas1 in proliferation, we speculated that Gas1 was connected to the activation of AMPK/mTOR/S6K axis and had an impact on the total and active states of AMPK/mTOR/S6K signaling pathway.

Forkhead box proteins are a family of transcription factors that play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, and metastasis (37). Recent studies demonstrated that FOXM1 plays an important role in regulating EMT (17, 18, 38, 39) and Warburg effect (40). Our data here demonstrated that the Gas1 promoter was sufficient to allow FOXM1-dependent inactivation of gene expression in transactivation assays and led to the identification of a bona fide FOXM1 responsive element within a 424 bp fragment of the Gas1 promoter, which contained a DNA sequence to which FOXM1 can directly bind. Negative correlation between FOXM1 and Gas1 was found in colorectal cancer cells and tissues. Collectively, our study indicated that Gas1 was transcriptionally regulated by FOXM1.

In summary, this study provided critical insight into the role of the Gas1 in colorectal cancer progression and identified that Gas1 played important roles by inhibiting both Warburg effect and EMT processes. Therefore, Gas1 may be a new biomarker and a therapeutic target for the treatment of colorectal cancer.

Acknowledgments

This research was supported by the National Science Foundation of China (Nos. 81372646 and 81101586) and National Key Basic Research Program of China (2014CBA02002). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).